Methods and systems for associating physical and genetic properties of biological particles

ABSTRACT

Provided herein are methods and systems for processing a nucleic acid molecule from a biological particle (e.g., cell). A plurality of partitions (e.g., droplets) may be generated such that partitions of the plurality of partitions each include a biological particle (e.g., cell) comprising the nucleic acid molecule and a particle (e.g., bead). The partitions can be processed (e.g., imaged) to obtain one or more physical and/or optical properties of their respective biological particles. The nucleic acid molecules included in the partitions can be barcoded and sequenced (e.g., using nucleic acid barcode molecules coupled to the particles of the partitions) to generate nucleic acid sequences of the nucleic acid molecules. The nucleic acid sequences can be electronically associated with the one or more optical properties of the biological particles.

CROSS REFERENCE

This application is a continuation of International Application No.PCT/US18/61629 filed Nov. 16, 2018, which claims the benefit of U.S.Provisional Application No. 62/587,634, filed Nov. 17, 2017, whichapplications are incorporated herein by reference.

BACKGROUND

Samples may be processed for various purposes, such as identification ofa type of sample moiety within the sample. The sample may be abiological sample. The biological samples may be processed for variouspurposes, such as detection of a disease (e.g., cancer) oridentification of a particular species. There are various approaches forprocessing samples, such as polymerase chain reaction (PCR) andsequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

Biological samples may be processed wherein the sample information(e.g., phenotypic information) may be lost using existing methods. Suchsample processing may not be useful for analyzing cell-to-cellvariations in the sample. Further, existing methods can suffer frominefficient sample preparation methods, such as time-consumingprocedures that may include multiple steps.

SUMMARY

Recognized herein is a need for the improved methods for samplepreparation for analyzing cell-to-cell variation (e.g., phenotypicvariation and sequence variation) in a biological sample.

In an aspect, the present disclosure provides a method for processing anucleic acid molecule of a biological particle, comprising: (a)providing (1) a plurality of partitions, wherein a partition of saidplurality of partitions comprises (i) a particle comprising a pluralityof nucleic acid barcode molecules, wherein a nucleic acid barcodemolecule of said plurality of nucleic acid barcode molecules comprises abarcode sequence, and (ii) a biological particle comprising said nucleicacid molecule, and (2) a first data set comprising data indicative ofone or more physical properties of said biological particle stored incomputer memory, which data is generated upon sensing said biologicalparticle; (b) using said nucleic acid barcode molecule of said pluralityof nucleic acid barcode molecules and said nucleic acid molecule of saidbiological particle to generate a barcoded nucleic acid molecule; (c)generating a second data set comprising data identifying a nucleic acidsequence of said barcoded nucleic acid molecule or a derivative thereof;and (d) using said first data set and said second data set to associatesaid one or more physical properties of said biological particle withsaid nucleic acid sequence.

In some embodiments, said one or more physical properties of saidbiological particle includes a size of said biological particle, a shapeof said biological particle, a surface marker on said biologicalparticle, an inclusion in said biological particle, a structure of anorganelle in said biological particle, a number of organelles in saidbiological particle, a secretion or excretion with respect to saidbiological particle, or a localization of an organelle in saidbiological particle.

In some embodiments, said biological particle comprises a plurality ofnucleic acid molecules. In some embodiments, said plurality of nucleicacid molecules comprises a plurality of ribonucleic acid molecules. Insome embodiments, said plurality of nucleic acid molecules comprises aplurality of deoxyribonucleic acid molecules.

In some embodiments, said nucleic acid molecule is a ribonucleic acidmolecule. In some embodiments, said nucleic acid molecule is adeoxyribonucleic acid molecule.

In some embodiments, said one or more physical properties comprisesphenotypic information of said biological particle.

In some embodiments, the method further comprises optically detectingsaid particle to generate a third data set comprising data indicative ofone or more physical properties of said particle or said plurality ofnucleic acid barcode molecules. In some embodiments, said one or morephysical properties of said particle comprises an optical property ofsaid particle. In some embodiments, said physical property of saidparticle comprises a size, a shape, a circularity, a hardness, or asymmetry of said particle or a component thereof. In some embodiments,said optical property of said particle comprises an absorbance, abirefringence, a color, a fluorescence characteristic, a luminosity, aphotosensitivity, a reflectivity, a refractive index, a scattering, or atransmittance of said particle or a component thereof.

In some embodiments, nucleic acid barcode molecules of said plurality ofnucleic acid barcode molecules comprise barcode sequences, which barcodesequences are identical.

In some embodiments, said plurality of partitions is a plurality ofdroplets. In some embodiments, said plurality of partitions is aplurality of wells.

In some embodiments, said particle comprises one or more opticalbarcodes. In some embodiments, said plurality of nucleic acid barcodemolecules of said particle comprise said one or more optical barcodes.In some embodiments, said one or more optical barcodes comprises afluorescent dye, a nanoparticle, a microparticle, or any combinationthereof. In some embodiments, said one or more optical barcodes has anassociated optical intensity or frequency that is distinct with respectto other optical barcodes of said plurality of partitions. In someembodiments, (a)-(d) are repeated for an additional partition among saidplurality of partitions, wherein said additional partition comprises (i)an additional particle comprising an additional plurality of nucleicacid barcode molecules, wherein a nucleic acid barcode molecule of saidadditional plurality of nucleic acid barcode molecules comprises anadditional barcode sequence different than said barcode sequence, and(ii) an additional biological particle comprising an additional nucleicacid molecule, wherein said partition and said additional partitionyield optical signals at different intensities or frequencies uponsensing. In some embodiments, said nanoparticle comprises a quantum dot.In some embodiments, said nanoparticle comprises a Janus particle.

In some embodiments, said plurality of nucleic acid barcode molecules isconfigured in a geometric structure. In some embodiments, said geometricstructure is a nucleic acid origami.

In some embodiments, said one or more physical properties of saidbiological particle are identified by imaging said partition using (i)bright field microscopy, (ii) fluorescence microscopy, (iii) phasecontrast microscopy, (iv) multispectral microscopy, or (v) polarizationmicroscopy.

In some embodiments, said particle is a bead. In some embodiments, saidbead is a gel bead. In some embodiments, said plurality of nucleic acidbarcode molecules is releasably attached to said gel bead. In someembodiments, said gel bead comprises a polyacrylamide polymer.

In some embodiments, said one or more physical properties are one ormore optical properties. In some embodiments, the method furthercomprises, subsequent to (a), imaging said partition to optically detectsaid biological particle, thereby identifying said one or more opticalproperties of said biological particle. In some embodiments, the methodfurther comprises, prior to (a), imaging said biological particle toidentify said one or more optical properties of said biologicalparticle. In some embodiments, said biological particle is imaged priorto providing said biological particle in said partition.

In some embodiments, said particle is coupled to another particlecomprising one or more optical barcodes.

In some embodiments, (a)-(d) are repeated for additional partitionsamong said plurality of partitions, wherein said additional partitionseach comprise (i) an additional plurality of nucleic acid barcodemolecules, wherein a nucleic acid barcode molecule of said additionalplurality of nucleic acid barcode molecules comprises an additionalbarcode sequence different than said barcode sequence, and (ii) anadditional biological particle comprising an additional nucleic acidmolecule, and wherein said partition and said additional partitionscomprise a combination of particles comprising molecular barcodes andparticles comprising optical barcodes, which combination is differentacross said partition and said additional partitions.

In some embodiments, (a)-(d) are repeated for additional partitionsamong said plurality of partitions, wherein said additional partitionseach comprise (i) an additional plurality of nucleic acid barcodemolecules, wherein a nucleic acid barcode molecule of said additionalplurality of nucleic acid barcode molecules comprises an additionalbarcode sequence different than said barcode sequence, and (ii) anadditional biological particle comprising an additional nucleic acidmolecule, which additional partitions comprises at least 1,000partitions. In some embodiments, said additional partitions comprise atleast 10,000 partitions. In some embodiments, said additional partitionscomprise at least 100,000 partitions. In some embodiments, saidadditional partitions comprise a plurality of particles comprisingnucleic acid barcode molecules comprising at least 1,000 barcodesequences, which at least 1,000 barcode sequences are different acrosssaid partition and said additional partitions. In some embodiments, saidadditional partitions comprises a plurality of particles comprisingnucleic acid barcode molecules comprising at least 10,000 barcodesequences, which at least 10,000 barcode sequences are different acrosssaid partition and said additional partitions. In some embodiments, saidadditional partitions comprises a plurality of particles comprisingnucleic acid barcode molecules comprising at least 100,000 barcodesequences, which at least 100,000 barcode sequences are different acrosssaid partition and said additional partitions.

In some embodiments, said biological particle is a cell.

In some embodiments, said biological particle comprises a cell, or oneor more components thereof, in a matrix. In some embodiments, saidmatrix is a polymeric matrix. In some embodiments, said matrix is a gelmatrix.

In some embodiments, the method further comprises associating a proteinof said biological particle with said one or more physical properties ofsaid biological particle with said nucleic acid sequence. In someembodiments, said protein is a cell surface protein.

In some embodiments, the method further comprises associating one ormore ribonucleic acid sequences or one or more deoxyribonucleic acid(DNA) sequences of said biological particle with said one or morephysical properties. In some embodiments, said one or more DNA sequencescomprise epigenetic information. In some embodiments, said one or moreDNA sequences comprise chromatin information.

In another aspect, the present disclosures provides a kit comprising aplurality of particles, which plurality of particles comprise (i) aplurality of nucleic acid barcode molecules coupled thereto, whereinsaid plurality of barcode molecules comprise a plurality of barcodesequences, and (ii) a plurality of optical barcodes, wherein saidplurality of optical barcodes comprise a plurality of optical codes,wherein each particle of said plurality of particles comprises (i) asubset of said plurality of nucleic acid barcode molecules coupledthereto and (ii) a subset of said plurality of optical barcodes, whereinbarcode sequences of subsets of said plurality of nucleic acid barcodemolecules differ across particles of said plurality of particles, andwherein optical codes of subsets of said plurality of optical barcodesdiffer across said particles.

In some embodiments, each subset of said subsets of said plurality ofoptical barcodes comprise a single optical barcode.

In some embodiments, each subset of said subsets of said plurality ofoptical barcodes comprise two or more optical barcodes.

In some embodiments, said plurality of optical barcodes confers opticalproperties to said plurality of particles. In some embodiments, saidoptical properties are selected from the group consisting of anabsorbance, a birefringence, a color, a fluorescence characteristic, aluminosity, a photosensitivity, a reflectivity, a refractive index, ascattering, or a transmittance of said particle or a component thereof.

In some embodiments, said plurality of optical barcodes comprises one ormore fluorescent dyes, nanoparticles, microparticles, or any combinationthereof. In some embodiments, said plurality of optical barcodescomprises a plurality of nanoparticles. In some embodiments, saidplurality of optical barcodes comprises a plurality of quantum dots. Insome embodiments, said plurality of optical barcodes comprises aplurality of Janus particles. In some embodiments, said plurality ofoptical barcodes comprises a plurality of fluorescent dyes. In someembodiments, said plurality of fluorescent dyes comprises between 2-10fluorescent dyes. In some embodiments, said plurality of fluorescentdyes comprises a plurality of fluorescent dyes having the same emissionwavelength and different intensities. In some embodiments, saidplurality of fluorescent dyes comprises at least 20 differentintensities.

In some embodiments, each subset of said subsets of said plurality ofoptical barcodes comprises different optical codes.

In some embodiments, each subset of said subsets of said plurality ofoptical barcodes comprises a different combination of optical codes.

In some embodiments, said plurality of optical barcodes has associatedoptical intensities or frequencies that are distinct with respect to oneanother.

In some embodiments, said plurality of optical barcodes comprises atleast 1,000 different optical codes. In some embodiments, said pluralityof optical barcodes comprises at least 10,000 different optical codes.In some embodiments, said plurality of optical barcodes comprises atleast 100,000 different optical codes.

In some embodiments, nucleic acid barcode molecules within a givensubset of said subsets of said plurality of nucleic acid barcodemolecules comprise identical barcode sequences.

In some embodiments, said plurality of barcode sequences comprises atleast 1,000 different barcode sequences. In some embodiments, saidplurality of barcode sequences comprises at least 10,000 differentbarcode sequences. In some embodiments, said plurality of barcodesequences comprises at least 100,000 different barcode sequences.

In some embodiments, said plurality of particles comprises at least10,000 particles. In some embodiments, said plurality of partitionscomprises at least 100,000 particles.

In some embodiments, said plurality of nucleic acid barcode moleculescomprise a plurality of functional sequences, which plurality offunctional sequences are configured to interact with a plurality oftarget molecules. In some embodiments, said plurality of targetmolecules comprises a plurality of deoxyribonucleic acid molecules. Insome embodiments, said plurality of target molecules comprises aplurality of ribonucleic acid molecules. In some embodiments, saidplurality of functional sequences is configured to capture saidplurality of target molecules.

In some embodiments, said plurality of particles comprises a pluralityof beads. In some embodiments, said plurality of particles comprises aplurality of gel beads. In some embodiments, said plurality of nucleicacid barcode molecules is releasably coupled to said plurality of gelbeads. In some embodiments, said plurality of gel beads comprises apolyacrylamide polymer.

In some embodiments, said plurality of optical barcodes is included onsurfaces of said plurality of particles.

In some embodiments, said plurality of optical barcodes is includedwithin said plurality of particles.

In some embodiments, first particles of said plurality of particles arecoupled to second particles of said plurality of particles, wherein saidfirst particles comprise nucleic acid barcode molecules of saidplurality of nucleic acid barcode molecules and said second particlescomprise optical barcodes of said plurality of optical barcodes.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 shows an example of a microfluidic channel structure for opticaldetection of partitions.

FIG. 9 shows an example of a procedure for electronically associatinggenetic and phenotypic information of biological particles.

FIG. 10 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. The subject can be a vertebrate, a mammal, a rodent (e.g., amouse), a primate, a simian or a human. Animals may include, but are notlimited to, farm animals, sport animals, and pets. A subject can be ahealthy or asymptomatic individual, an individual that has or issuspected of having a disease (e.g., cancer) or a pre-disposition to thedisease, and/or an individual that is in need of therapy or suspected ofneeding therapy. A subject can be a patient.

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Cross-linking can be via covalent, ionic, or inductive,interactions, or physical entanglement. The bead may be a macromolecule.The bead may be formed of nucleic acid molecules bound together. Thebead may be formed via covalent or non-covalent assembly of molecules(e.g., macromolecules), such as monomers or polymers. Such polymers ormonomers may be natural or synthetic. Such polymers or monomers may beor include, for example, nucleic acid molecules (e.g., DNA or RNA). Thebead may be formed of a polymeric material. The bead may be magnetic ornon-magnetic. The bead may be rigid. The bead may be flexible and/orcompressible. The bead may be disruptable or dissolvable. The bead maybe a solid particle (e.g., a metal-based particle including but notlimited to iron oxide, gold or silver) covered with a coating comprisingone or more polymers. Such coating may be disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The biologicalsample may be a nucleic acid sample or protein sample. The biologicalsample may also be a carbohydrate sample or a lipid sample. Thebiological sample may be derived from another sample. The sample may bea tissue sample, such as a biopsy, core biopsy, needle aspirate, or fineneedle aspirate. The sample may be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample may be a skin sample.The sample may be a cheek swab. The sample may be a plasma or serumsample. The sample may be a cell-free or cell free sample. A cell-freesample may include extracellular polynucleotides. Extracellularpolynucleotides may be isolated from a bodily sample that may beselected from the group consisting of blood, plasma, serum, urine,saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a virus. The biological particle may be acell or derivative of a cell. The biological particle may be anorganelle. The biological particle may be a rare cell from a populationof cells. The biological particle may be any type of cell, includingwithout limitation prokaryotic cells, eukaryotic cells, bacterial,fungal, plant, mammalian, or other animal cell type, mycoplasmas, normaltissue cells, tumor cells, or any other cell type, whether derived fromsingle cell or multicellular organisms. The biological particle may beor may include a matrix (e.g., a gel or polymer matrix) comprising acell or one or more constituents from a cell (e.g., cell bead), such asDNA, RNA, organelles, proteins, or any combination thereof, from thecell. The biological particle may be obtained from a tissue of asubject. The biological particle may be a hardened cell. Such hardenedcell may or may not include a cell wall or cell membrane. The biologicalparticle may include one or more constituents of a cell, but may notinclude other constituents of the cell. An example of such constituentsis a nucleus or an organelle. A cell may be a live cell. The live cellmay be capable of being cultured, for example, being cultured whenenclosed in a gel or polymer matrix, or cultured when comprising a gelor polymer matrix.

The biological particle may be a fixed cell or a population of fixedcells, such as a tissue. The biological particle may be FFPE cells ortissues. The biological particle may be obtained from an FFPE sample bylaser capturing the cells.

A cell may split into two or more daughter cells, such as by binaryfission, for example. The cell can be at any stage of cell division(e.g., prophase, metaphase). One or more physical properties of thebiological particle can include a number of daughter cells, stage ofcell division, and/or type of cell division.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. Themacromolecular constituent may comprise DNA. The macromolecularconstituent may comprise RNA. The RNA may be coding or non-coding. TheRNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA(tRNA), for example. The RNA may be a transcript. The RNA may small RNAthat are less than 200 nucleic acid bases in length, or large RNA thatare greater than 200 nucleic acid bases in length. Small RNAs mainlyinclude 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA),microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA(snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA)and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNAor single-stranded RNA. The RNA may be circular RNA. The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. The partition may isolate space or volume fromanother space or volume. The partition may be a droplet or well, forexample. The droplet may be a first phase (e.g., aqueous phase) in asecond phase (e.g., oil) immiscible with the first phase. The dropletmay be a first phase in a second phase that does not phase separate fromthe first phase, such as, for example, a capsule or liposome in anaqueous phase.

The present disclosure provides methods and systems for obtaininggenetic and/or proteomic information from a biological particle (e.g., acell) and associating such information with one or more physicalproperties of the biological particle, such as size, shape, or density,or any other phenotypic property of the biological particle. This maypermit genetic and/or proteomic information from the biological particleto be linked to the one or more physical properties of the biologicalparticle.

Methods and Systems for Associating Physical and Genetic Properties ofBiological Particles

In an aspect, the present disclosure provides a method for processing anucleic acid molecule of a biological particle (e.g., a cell). Themethod may comprise providing a plurality of partitions (e.g., aplurality of wells or droplets). A partition of the plurality ofpartitions may comprise (i) a particle comprising a plurality of nucleicacid barcode molecules; and (ii) a biological particle comprising thenucleic acid molecule. A nucleic acid barcode molecule of the pluralityof nucleic acid barcode molecules may comprise a barcode sequence. Themethod may further comprise providing a first data set comprising dataindicative of one or more physical properties of the biologicalparticle. The first data set may be stored in computer memory. The firstdata set may be generated upon sensing the biological particle, forexample. The one or more physical properties may include, for example, asize, shape, or density of the biological particle. The sensing of theone or more physical properties may generate optical information, suchas by optically detecting the biological particle and/or the partition.

A given nucleic acid barcode molecule of the plurality of nucleic acidbarcode molecules may be used to barcode the nucleic acid molecule ofthe biological particle (e.g., included within or derived from thebiological particle), to generate a barcoded nucleic acid molecule. Asecond data set comprising data identifying a nucleic acid sequence ofthe barcoded nucleic acid molecule or a derivative thereof may then begenerated (e.g., using nucleic acid sequencing). The first data set andthe second data set may then be used to associate (e.g., electronicallyassociate) the one or more physical properties of the biologicalparticle with the nucleic acid sequence.

The biological particle can be a cell. As an alternative, the biologicalparticle can be a gel or polymer matrix comprising the cell, aderivative of the cell, or one or more constituents of the cell.

In some examples, the biological particle is a cell. The cell may splitinto two or more daughter cells, such as by binary fission, for example.The cell can be at any stage of cell division (e.g., prophase,metaphase). The one or more physical properties of the cell can includea number of daughter cells, stage of cell division, and/or type of celldivision. The biological particle may comprise a plurality of nucleicacid molecules. Some or all of the nucleic acid molecules may bedisposed in the interior of the cell. Alternatively, the nucleic acidmolecules may be disposed external or partially external to the cell.The cell may be lysed or permeabilized to provide access to one or morenucleic acid molecules within the cell.

One or more nucleic acid molecules of or associated with the biologicalparticle may be associated with the one or more physical properties ofthe biological particle. For example, at least 1, 10, 100, 1,000,10,000, 100,000 or 1,000,000 nucleic acid molecules from or associatedwith the biological particle may be associated with the one or morephysical properties of the biological particle.

One or more nucleic acid molecules may be conjugated to one or moreantibodies coupled to one or more proteins coupled to a surface of thebiological particle (e.g., cell). Identifying a sequence of such one ormore nucleic acid molecules may permit identifying the one or moreantibodies and the one or more proteins. The one or more proteins fromthe biological particle may subsequently be associated with the one ormore physical properties of the biological particle. Such proteomicinformation may be used in association with genetic information from thebiological particle, such as DNA sequence information, transcriptomeinformation (i.e., sequences of transcripts), or both. For example, acell surface protein of a cell can be identified using one or moreantibodies. The cell surface protein can be associated with one or morephysical properties of the cell (e.g., a shape of the cell) with anucleic acid molecule. The one or more physical properties can becharacterized by imaging the cell. The nucleic acid molecule of aderivative thereof of the cell can be sequenced to obtain a nucleic acidsequence. The nucleic acid sequence can be associated with the cellsurface protein, in turn, with the one or more physical properties ofthe cell (e.g., a shape of the cell). In some cases, one or moreribonucleic acid (RNA) sequences and/or one or more deoxyribonucleicacid (DNA) sequences of a biological particle (e.g., cell) can beassociated with one or more physical properties. In some cases, the oneor more DNA sequences can comprise epigenetic information. For example,the one or more DNA sequences can include methylated DNA (e.g.,5-methylcytosine) as indicated by an epigenetic assay (e.g., bisulfitesequencing). In some cases, the one or more DNA sequences can includechromatin information, such as a lower nucleosome occupancy resulting ina highly accessible chromatin or “open” chromatin. The open chromatincan be as assessed by subjecting the one or more DNA sequences to anenzymatic treatment (e.g., DNase I, transposase), releasing a segment ofnucleic acid molecules between two nucleosomes in an open chromatin.

A partition (e.g., droplet) can comprise a particle (e.g., bead) and abiological particle (e.g., cell). The partition can be distinguishedfrom other partitions in order to uniquely identify the partition. Thepartition can be characterized by, for example, imaging the partition toobtain optical information of the particle, a plurality of nucleic acidbarcode molecules attached to the particle and/or the biologicalparticle. In an example, the partition may be distinguished from otherpartitions by the particle included in the partition. For example, theparticle may include one or more features such as one or more opticalbarcodes or other characteristics that may be used to distinguish thepartition from other partitions. A lookup table (LUT) can be used toassociate the plurality of nucleic acid barcode molecules to theparticle. The optical information of the partition can permitassociating the particle (e.g., gel bead) with the biological particle(e.g., cell) in the partition (e.g., droplet). The association of theparticle with the biological particle can further permit associating anucleic acid sequence of a nucleic acid molecule of the biologicalparticle to one or more physical properties (e.g., a color of a cell) ofthe biological particle. For example, the partition (e.g., droplet) caninclude a particle (e.g., bead) having a first color and a biologicalparticle (e.g., cell) having a second color or other opticalcharacteristic, such as a shape or morphology of the biologicalparticle, which first color and second color or characteristic can beassociated with one another. The particle can have a plurality ofnucleic acid barcode molecules attached thereto. The plurality ofnucleic acid barcode molecules can comprise barcode sequences. Theplurality of nucleic acid molecules attached to a given particle canhave the same barcode sequence.

In some examples, the LUT can be used to associate a feature (e.g., anoptical barcode, such as a color and/or intensity) of the particle withthe barcode sequence. The feature may derive from the particle or anoptical tag associated with the particle. The partition (e.g., droplet)can be imaged to obtain optical information of the partition, including,for example, the feature (e.g., color and/or intensity) of the particleor the optical tag associated with the particle, and optical informationof the biological particle in the partition. For example, an image caninclude optical information in the visible spectrum, non-visiblespectrum, or both. For example, multiple images may be obtained of apartition across various optical frequencies.

The biological particle can comprise a nucleic acid molecule which canbe barcoded with a barcode sequence of a nucleic acid barcode moleculeof the particle in the partition to provide a barcoded nucleic acidmolecule. The barcoded nucleic acid molecule can be sequenced to obtaina nucleic acid sequence. The nucleic acid sequence can comprise geneticinformation of the biological particle. The nucleic acid sequence maycomprise the barcode sequence, or a complement thereof. The barcodesequence, or complement thereof, of the nucleic acid sequence can beelectronically associated with the feature (e.g., color and/orintensity) of the particle using the LUT to identify the particle.Alternatively, the LUT may be used to look up the barcode sequence toidentify the feature (e.g., color and/or intensity), which maysubsequently be used to identify an image having such feature, which mayidentify the partition comprising the particle. Identification of theparticle can be useful in identifying the partition. This may in turnpermit identification of the biological particle by using the opticalinformation of the partition. This may permit genotypic information froma biological particle (e.g., cell) to be associated with phenotypicinformation of the biological particle.

In an example, an image of a partition (e.g., droplet) comprising abiological particle (e.g., cell or cell bead) and a particle (e.g.,bead) comprising nucleic acid barcode molecules is captured. The nucleicacid barcode molecules include the same barcode sequence. The imageidentifies the particle as having a color (e.g., a green color) and alsocaptures a shape of the biological particle (e.g., cell). The nucleicacid barcode molecules are used to barcode a nucleic acid molecule(e.g., an RNA molecule) from the biological particle (e.g., cell) togenerate a sequencing library that is subsequently sequenced to yield aplurality of sequencing reads. The some or all of the plurality ofsequencing reads include the barcode sequence. Next, an LUT is used toassociate the color of the particle from the captured image to thebarcode sequence, which permits sequencing reads generated from thesequencing library to be identified. The sequencing reads are thenassociated with the shape of the biological particle.

In some cases, one or more optical properties of the partition (e.g.,droplet) and/or components (e.g., particle or biological particle)thereof can be used to distinguish the individual partition from otherpartitions. In some cases, the optical properties of the partition, suchas color of a partitioning fluid (e.g., aqueous fluid) can be used todistinguish the partition from other partitions. For example, thepartitions can have different colors associated therewith to be able todistinguish from the partitions from one another. In some cases, thepartitions can include droplets having different colors associatedtherewith. The partition can be any container or a vessel, such as awell, microwell, tube, nanoarrays or other containers. In some cases,the plurality of partitions can include at least 1,000 partitions, atleast 10,000 partitions or at least 100,000 partitions. In such cases,at least a portion of the plurality of partitions can have one or moreunique optical properties. In an example, one or more optical propertiesof the partition (e.g., droplet) and/or components (e.g., particle orbiological particle) thereof may be used to identify whether thepartition includes a particle and/or biological particle or not (e.g.,whether the partition is occupied or unoccupied).

Non-limiting examples of optical properties of a partition or acomponent thereof (e.g., particle or biological particle) can includeabsorbance, birefringence, color, fluorescence, luminosity,photosensitivity, reflectivity, refractive index, scattering, ortransmittance. Such optical properties may be associated with thepartition, particle, or biological particle, such as associated with asignal directly from the partition, particle, or biological particle, orfrom one or more moieties coupled to the partition, particle, orbiological particle.

The partitions and/or components thereof (e.g., particles or biologicalparticles) can be made optically detectable by including an opticalmaterial in the partition to distinguish the partitions from oneanother. For example, the plurality of partitions can comprise a firstpartition and a second partition. The first partition and the secondpartition can yield optical signals at different intensities and/orfrequencies. For example, the first and second partitions can includeone or more optical materials such as one or more fluorescent dyes,nanoparticles, and/or microparticles that can provide a distinct opticalsignal. The one or more optical materials may be associated with thepartition (e.g., a partitioning fluid), the particle (e.g., nucleic acidbarcode molecules of the particle), or the biological particle. In someexamples, the partitioning fluid (e.g., aqueous fluid) can include afluorescent dye. In some examples, the partitioning fluid can includenanoparticles, such as quantum dots, to provide enhanced spectralresolution in order to distinguish between the partitions. In somecases, the one or more optical materials can be introduced prior to orsubsequent to generating partitions. For example, a fluorescent dye canbe injected into the partitioning fluid after generating partitions(e.g., droplets). In some examples, a fluorescent dye can be included inthe partitioning fluid before generating partitions (e.g., droplets).

In some cases, optical properties of the particles (e.g., beads) can beused for optical detection of the particles (e.g., within thepartitions). For example, the particles can have different colors to beable to distinguish from one another. In some examples, the particlescan have different refractive indices based on the composition of theparticles. The particles may be beads such as gel beads. Gel beads maycomprise a polyacrylamide polymer. Gel beads may have differentbirefringence values based on degree of polymerization, chain length, ormonomer chemistry. The particle can include (e.g., encapsulate or haveattached thereto) a plurality of nucleic acid molecules, which can benucleic acid barcode molecules. In some cases, optical properties of thenucleic acid barcode molecules can be used for optical detection of theparticles. For example, the absorbance of light by the nucleic acidbarcode molecules can be used to distinguish the particles from oneanother.

The particles can be made optically detectable by including opticalbarcodes in the particles, on surfaces of the particles, or both in andon surfaces of the particles. The optical barcodes can be on interiorand/or exterior surfaces of the particles. Optical barcodes may compriseoptical codes (e.g., one or more characteristic frequencies and/orintensities). Non-limiting examples of the optical barcodes can includefluorescent dyes, nanoparticles, and/or microparticles. For example, theparticles can include a plurality of different fluorescent dyes.Fluorescent dyes may be attached to the surface of the particles and/ormay be incorporated into the particles for use as optical barcodes orcomponents thereof. Fluorescent dyes may be attached to a surface ofparticles, incorporated within particles, and/or attached to nucleicacid barcode molecules coupled to particles. Different intensities ofthe different fluorescent dyes may be used to increase the number ofoptical barcodes (e.g., optical codes) that may be used. For example, ifN is the number of fluorescent dyes (e.g., between 2 and 10 fluorescentdyes, such as 4 fluorescent dyes) and M is the possible intensities forthe dyes (e.g., between 2 and 50 intensities, such as 20 intensities),then M^(N) are the possible distinct optical barcodes. In one example, 4fluorescent dyes with 20 possible intensities will be used to generate160,000 distinct optical barcodes. In some examples, nanoparticles, suchas quantum dots or Janus particles, can be used as optical barcodes orcomponents thereof. In some examples, the quantum dots can be attachedto nucleic acid barcode molecules of the particles. Optical barcodes(e.g., optical codes) of particles may provide enhanced spectralresolution in order to distinguish between particles with unique nucleicacid barcode molecules (e.g., particles comprising unique barcodesequences). In an example, a first particle comprises a first opticalbarcode and nucleic acid barcode molecules each having a first barcodesequence. A second particle comprises a second optical barcode andnucleic acid barcode molecules each having a second barcode sequence.The first optical barcode and second optical barcode may be different(e.g., provided by two different fluorescent dyes or the samefluorescent dye at two different intensities). The first and secondbarcode sequences may be different nucleic acid sequences. The particlesmay be imaged to sense the first and second optical barcodes and thefirst and second optical barcodes may then be used to associate thefirst and second optical barcodes with the first and second barcodesequences, respectively.

Optical barcodes may be included while generating the particles. Forexample, the particles can be beads (e.g., gel beads). Gel beads cancomprise polyacrylamide polymers. Optical barcodes can be included inthe polymer structure, or attached at the pre-polymer or monomer stagein bead production. In some cases, the beads may comprise moieties thatcan attach to one or more optical barcodes (e.g., at a surface of a beadand/or within a bead). In some cases, optical barcodes can be loadedinto the beads with one or more reagents. For example, reagents andoptical barcodes can be loaded into the beads by diffusion of thereagents (e.g., a solution of reagents comprising the optical barcodes).In some cases, optical barcodes can be included while preparing nucleicacid barcode molecules. For example, nucleic acid barcode molecules canbe prepared by synthesizing molecules comprising barcode sequences(e.g., using a split pool or combinatorial approach). Optical barcodesmay be attached to nucleic acid barcode molecules prior to attaching thenucleic acid barcode molecules to a particle. In some cases, opticalbarcodes can be included after attaching nucleic acid barcode moleculesto a particle. For example, optical barcodes can be attached to nucleicacid barcode molecules coupled to the particle. Nucleic acid barcodemolecules or sequences thereof can be releasably attached to theparticle. Optical barcodes can be releasably or non-releasably attachedto the particle. In some cases, a first particle (e.g., a particlecomprising a plurality of nucleic acid barcode molecules) can be coupledto a second particle comprising one or more optical barcodes. Forexample, the first particle can be covalently coupled to the secondparticle via a chemical bond. In some cases, the first particle can benon-covalently associated with the second particle. The first and/orsecond particle may comprise a plurality of nucleic acid barcodemolecules. The plurality of nucleic acid barcode molecules coupled to agiven particle can comprise the same barcode sequences. Where both thefirst and second particles comprise nucleic acid barcode molecules, thefirst and second particles may comprise nucleic acid barcode moleculescomprising the same barcode sequences or different barcode sequences.

Optical properties of a biological particles (e.g., cell) can be usedfor optical detection of the biological particles. The optical detectionof the biological particles can provide one or more physical properties(e.g., optical information) of the biological particle. The one or morephysical properties of the biological particle can include phenotypicinformation. The phenotypic information of the biological particle caninclude one or more members selected from the group consisting of a sizeof the biological particle, a shape of the biological particle, asurface marker on the biological particle, an inclusion in thebiological particle, a structure of an organelle in the biologicalparticle, number of organelles in the biological particle, secretion orexcretion with respect to the biological particle, a localization of anorganelle in the biological particle, a circularity of the biologicalparticle, a density of the biological particle, symmetry of thebiological particle, and hardness of the biological particle. Opticalproperties comprising or correlated with phenotypic information can beused to characterize the biological particle. For example, absorbance ofan inclusion in the cell (e.g., pigment granules) can be used todistinguish biological particles from one another. In some cases, abiological particle can include one or more components of a cell in amatrix (e.g., polymeric matrix or a gel matrix). In such cases, opticalproperties of the matrix can be used for optical detection of abiological particle (e.g., cell) or components thereof included in thematrix. In some cases, phenotypic information of the biological particle(e.g., cell) can be preserved by encapsulating (e.g., in microcapsule)the biological particle prior to partitioning.

Biological particles can be made optically detectable by includingoptical materials (e.g., optical barcodes or labels) in the biologicalparticles, on surfaces of the biological particles, or both in and onsurfaces of the biological particles. Optical materials can be on theinterior and or exterior surfaces (e.g., cell membranes) of biologicalparticles. For example, biological particles (e.g., cells) or thecomponents thereof (e.g., nucleic acid molecule) can be labelled withfluorescent dyes. In some examples, nucleic acid molecules, such as DNAmolecules, can be fluorescently labelled using4′,6-diamidino-2-phenylindole (DAPI). The fluorescence from binding ofDAPI to DNA can be imaged to optically detect the biological particle.In some cases, optical materials can be attached to antibodies. Forexample, a biological particle or components thereof can be opticallydetected using optical materials (e.g., fluorescent dyes such as AlexaFluor) conjugated to an antibody against a protein. In some cases,biological particles can be engineered to express one or more opticalmaterials. For example, a biological particle (e.g., cell) can beengineered to express Green Fluorescent Protein (GFP).

One or more physical properties of a partition (e.g., droplet) and/orcomponents (e.g., particle and/or biological particle) thereof can beused to distinguish a partition from other partitions. Non-limitingexamples of the physical properties can include size, shape,circularity, hardness, volume, or symmetry of the partition and/orcomponents thereof. In some cases, physical properties of a partitioncan be used to distinguish the partition from other partitions. Forexamples, the volume of the partitioning fluid (e.g., aqueous fluid) canbe used to distinguish the partition from other partitions. In otherexamples, the shape and/or the symmetry of the partition (which mayreflect characteristics of the particle and/or biological particleincluded therein) can be used to distinguish the partition form otherpartitions. Partitions can be made physically detectable by generatingpartitions with unique physical properties. In some cases, the pluralityof partitions can include at least 1,000 partitions, at least 10,000partitions or at least 100,000 partitions. In such cases, at least aportion of the partitions can have one or more unique physicalproperties. For example, the partitions can be generated with differentvolumes. In some cases, the volume of partitioning fluids (e.g., aqueousfluid) can be different in the plurality of partitions.

Physical properties of particles (e.g., beads) can be used tocharacterize the particles (e.g., within partitions). The particles canbe beads, such as gel beads. The particles can be biological particles.The particles can include (e.g., encapsulate or have attached thereto)nucleic acid molecules, which can be nucleic acid barcode molecules. Thenucleic acid barcode molecules can include barcode sequences (e.g.,nucleic acid barcode sequences). The barcode sequences can be the sameacross a plurality of the nucleic acid barcode molecules (e.g., aplurality of nucleic acid barcode molecules coupled to a given bead).Non-limiting examples of physical properties of particles can includesize, shape, circularity, density, symmetry, and hardness. For example,particles can be of different sizes. Different sizes of particles can beobtained by using microfluidic channel networks configured to providespecific sized particles (e.g., based on channel sizes, flow rates,etc.). In some examples, particles can have different hardness valuesthat can be obtained by varying the concentration of polymer used togenerate the particles. In some cases, a nucleic acid barcode moleculeattached to a particle (e.g., bead) can be made optically detectableusing a physical property of the nucleic acid molecule. For example, anucleic acid origami, such as a deoxyribonucleic acid (DNA) origami canbe used to generate an optically detectable nucleic acid barcodemolecule. For example, a nucleic acid molecule, or a plurality ofnucleic acid molecules, can be folded to create two-and/orthree-dimensional geometric shapes. The different geometric shapes canthen be further optically detected. In some examples, special types ofnanoparticles with more than one distinct physical property can be usedto make the particles physically distinguishable. In an example, Janusparticles with both hydrophilic and hydrophobic surfaces can be used toprovide unique physical property. In some cases, physical properties ofparticles can be characterized prior to and/or subsequent topartitioning.

A partition can be characterized using one or more physical and/oroptical properties of the partitions and/or components thereof (e.g.,particles, nucleic acid barcode molecules of particles, and/orbiological particles) by imaging the partition. The partition can beimaged using an imaging unit. The imaging unit can be configured toimage the partition based on an expected property (e.g., opticalproperty) of the partition to be characterized. For example, an imagingunit can be configured to excite and subsequently detect an emissionwavelength of a partition comprising one or more fluorescent dyes. Theimaging unit can be configured to perform bright field microscopy,fluorescence microscopy, phase contrast microscopy, multispectralmicroscopy, or polarization microscopy. The imaging unit can beconfigured to include an optical sensor based on the microscopyperformed. In some examples, the imaging unit can be configured toperform bright field microscopy to determine the physical property ofthe partition (e.g., shape and/or size of the partition, particle andthe biological particle). In other examples, the imaging unit can beconfigured to perform fluorescence microscopy to image the partition(e.g., fluorescently-labelled partition, particle and/or biologicalparticle). In some cases, microfluidic chips can include additionalfeatures for imaging the individual partitions without interference. Forexample, the microfluidic chip can include features, such as rails,asymmetric turns, herringbone mixers, to facilitate the imaging of thepartitions without interference.

As illustrated in FIG. 8, the microfluidic channel structure 800 caninclude the channel segments 801, 802, 804, 806, and 808 communicatingat a channel junction 810. In operation, the channel segment 801 cantransport an aqueous fluid or a first liquid phase that includes aplurality of beads 812 (e.g., with nucleic acid barcode molecule), andreagent 814 along the channel segment 801. The plurality of beads 812can be releasably attached to a plurality of nucleic acid barcodemolecules with barcode sequences and/or additional sequences (e.g., R1and R2). The plurality of beads 812 may be sourced from a suspension ofbeads. For example, the channel segment 801 may be connected to areservoir comprising an aqueous suspension of beads. The reagent 814 caninclude a lysis reagent, PCR reagents, ligase etc. The channel segment802 may transport the aqueous fluid that includes a plurality ofbiological particles 816 along the channel segment 802 into junction810. The plurality of biological particles 816 may be sourced from asuspension of biological particles. The plurality of biologicalparticles 816 can be cells or derivate thereof, such as macromolecularconstituents. The plurality of biological particles 816 can be a sampleobtained from formalin-fixed paraffin embedded tissues. A second fluidor a second liquid phase 818 that is immiscible with the aqueous fluid(e.g., oil) can be delivered to the junction 810 via channel segment804. Upon meeting of the aqueous fluid from each of channel segments 801and 802 and the second fluid 818 from the channel segment 804 at thechannel junction 810, the aqueous fluid can be partitioned as discretedroplets 816 in the second fluid 814 and flow away from the junction 810along channel segment 808. The channel segment 808 may deliver thediscrete droplets to an outlet reservoir fluidly coupled to the channelsegment 808, where they may be harvested. An individual droplet 820 mayinclude a biological particle, a particle and reagents. The individualdroplet 820 can be imaged with an imaging unit 822. The imaging unit 822can be communicatively coupled to the microfluidic channel structure800. The imaging unit 822 can be configured to image the individualdroplet as the droplet flows through the channel segment 808. Theimaging unit 822 can provide optical information of the droplet to acontroller 824. The controller 822 can be operatively coupled to theimaging unit 822. The controller 824 can be configured to direct fluidflow in the channel segment 808. The controller 824 can be configured tocommunicate the optical information to a processor 826 for furtherprocessing. As an alternative to the imaging unit 822 or in addition tothe imaging unit 822, other sensors may be used to detect one or morephysical properties of the individual droplet 820 and/or constituents ofthe individual droplet 820.

In some cases, more than one property of and/or from the partition canbe assessed. For example, size of the particle and optical barcodes onthe particle can be imaged using the imaging unit. In some cases, thepartition can be imaged prior to injecting the particle in thepartition. For example, the biological particle (e.g., cell, cell beador microcapsule) can be imaged prior to injecting the particle (e.g.,bead). The biological particle can be imaged prior to and subsequent topartitioning into the partition (e.g., droplet). In another example, thepartition can be imaged after injecting the particle in the partition.For example, the partition comprising the biological particle and theparticle can be imaged. Upon imaging the partition, optical informationcan be generated. The optical information can include opticalinformation of the partition and/or particle. In some cases, the opticalinformation can include optical information of the particle (e.g.,bead). The optical information can include phenotypic information of thebiological particle (e.g., cell). In some cases, the optical informationcan include the optical information of the particle and the phenotypicinformation of the biological particle.

Upon imaging, the individual partitions can carry out reactions togenerate a barcoded nucleic acid molecule. The biological particle cancomprise a nucleic acid molecule. The biological particle can comprise aplurality of nucleic acid molecules. The nucleic acid molecule(s) cancomprise ribonucleic acid (RNA). The nucleic acid molecule(s) cancomprise deoxyribonucleic acid (DNA). The biological particle can be asingle biological particle, such as a cell or a polymer matrix (e.g.,cell bead) as disclosed elsewhere herein. The biological particle can bea plurality of biological particles, such as a plurality of cells. Thebiological particle can comprise a live or fixed cell, such as FFPEcells. The biological particle can be any cellular derivative orcombination thereof, such as DNA, RNA, nucleus, etc. The biologicalparticle can be encapsulated in a gel matrix to preserve the particle.

The biological particle can be encapsulated prior to and/or subsequentto partitioning into a partition. The partition can be any container ora vessel, such as a well, microwell, tube, nanoarrays or othercontainers. The partition can be flowable within fluid streams, such asa microcapsule having an inner fluid core surrounded by an outerbarrier. The partition can be a droplet of aqueous fluid within anon-aqueous phase, such as an oil phase. Partitioning one or morematerials (e.g., a particle and a biological particle) into a partitioncan be performed by any of the methods disclosed herein.

The particle can comprise a plurality of nucleic acid barcode molecules.The plurality of nucleic acid barcode molecules can comprise barcodesequences (e.g., nucleic acid barcode sequences). In some cases, thebarcodes sequences can be the same across a plurality of nucleic acidbarcode molecules. The plurality of partitions can comprise a pluralityof particles comprising a plurality of nucleic acid barcode molecules.The plurality of nucleic acid barcode molecules can comprise at least1,000 barcode sequences which at least 1,000 barcode sequences, at least10,000 barcode sequences or at least 100,000 barcode sequences can bedifferent across the plurality of partitions. In some cases, the nucleicacid barcode molecule can comprise additional sequences. The additionalsequences can include a primer binding site, such as a sequencing primersite (e.g., R1 or R2), flow cell binding sequences (e.g., P5, P7). Aprimer binding site can comprise sequences for sequencing primers tohybridize with a nucleic acid in a sequencing reaction. A primer bindingsite can comprise sequences for primers to hybridize with a nucleic acidin amplification or other extension reactions. The additional sequencescan be useful in downstream assays, such as a sequencing assay. Theadditional sequences can be selected based on the assay used.

Once released from the bead, a give nucleic acid barcode molecule orportion thereof of a plurality of the nucleic acid molecules coupled toa particle can barcode the nucleic acid molecule from the biologicalparticle to generate a barcoded nucleic acid molecule. The given nucleicacid barcode molecule can attach to the nucleic acid molecule byannealing, extension and amplification reaction and/or ligationreactions. Extension and amplification reagents, such as DNA polymerase,nucleoside triphosphates, and buffers with co-factors (e.g. Mg²⁺), canbe co-partitioned with biological particles and beads. The nucleic acidbarcode molecule can be attached at either one or both ends of thesegment to yield a barcoded nucleic acid molecule. Alternatively or inaddition, a nucleic acid hybridization and extension process may takeplace within a partition and additional extension or amplificationreactions may take place outside a partition (e.g., upon poolingmaterials from a plurality of such partitions).

Barcoded nucleic acid molecules or derivatives thereof (e.g., barcodednucleic acid molecules to which one or more functional sequences havebeen added, or from which one or more features have been removed) ofdifferent partitions can be pooled and processed together for subsequentanalysis such as sequencing on high throughput sequencers. Processingwith pooling may be implemented using barcode sequences. For example,barcoded nucleic acid molecules of a given partition may have the samebarcode sequence, which barcode sequence is different from barcodesequences of other partitions. Alternatively, barcoded nucleic acidmolecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing). Sequencing of barcoded nucleicacid molecules (e.g., in a pooled mixture or separately) can providesequencing reads comprising nucleic acid sequences. Such nucleic acidsequences may comprise the barcode sequences of the barcoded nucleicacid molecules, or complements thereof. For example, a plurality ofsequencing reads corresponding to a given partition (e.g., a givenbarcoded nucleic acid molecule or collection thereof) may be generated,in which a subset of the plurality of sequencing reads comprises thebarcode sequence of the barcoded nucleic acid molecule or a complementthereof. The nucleic acid sequence may comprise a sequence correspondingto the nucleic acid barcode molecules of a particle in a partitionand/or a sequence corresponding to the nucleic acid molecule of abiological particle in the partition.

A barcode sequence in a nucleic acid sequence can enable electronicassociation of physical and/or optical information of the biologicalparticle (e.g., cell) from which a nucleic acid molecule (and thusbarcoded nucleic acid molecule) was derived with the nucleic acidsequence. For example, a barcode sequence in a nucleic acid sequence canenable the identification of the particle from which the barcodesequence of the nucleic acid barcode molecule was derived. Theidentification of the particle can enable identification of thepartition by electronically associating physical and/or opticalinformation of the particle with the partition. The identification ofthe partition can enable identification of the biological particle inthe partition by electronically associating the optical information ofthe biological particle (e.g., cell) with the partition. Theidentification of the biological particle can enable associating anyphenotypic information (e.g., cell size, shape, etc.) associated withthe biological particle with the nucleic acid sequence. In some cases,the electronic association of the phenotypic information of thebiological particle with the nucleic acid sequence may be useful inassaying cell response to a therapy. For example, the response to thetherapy may include changes in phenotypic information of the cell (e.g.,number of organelles). The changes in phenotypic information of the cellcan be associated with the nucleic acid sequence of the nucleic acidmolecule of the cell.

As illustrated in FIG. 9, a plurality of partitions can include a firstpartition 902 and a second partition 904. A first partition 902 caninclude a first biological particle (e.g., cell) 906 and a firstparticle (e.g., gel bead) 908. A second partition 904 can include asecond biological particle (e.g., cell) 910 and a second particle (e.g.,gel bead) 912. The first partition 902 and the second partition 904 caneach have a unique physical and/or optical property for differentiatingfrom other partitions. The unique physical and/or optical property maybe a property of the partition itself (e.g., a volume or other featureof the partition) and/or a property of a component of the partition,such as a particle or biological particle of the partition. For example,the first particle 908 can be a round bead and the second particle 912can be a square bead. The first and second particles can comprise aplurality of nucleic acid barcode molecules coupled thereto. Theplurality of nucleic acid barcode molecules can comprise barcodesequences. The barcode sequences of nucleic acid barcode moleculescoupled to a given particle (e.g., the first particle) can be the same(e.g., nucleic acid barcode molecules coupled to the first particle eachcomprise the same barcode sequence and nucleic acid barcode moleculescoupled to the second particle each comprise the same barcode sequence),while the barcode sequences associated with the first particle may bedifferent from the barcode sequences associated with the secondparticle. For example, the first particle can have a first nucleic acidbarcode molecule comprising a first barcode sequence 914 and the secondparticle can have a second nucleic acid barcode molecule comprising asecond barcode sequence 916. The first barcode sequence of the firstnucleic acid barcode molecule 914 can be coupled to the first particle908 before and/or after partitioning the particle. The second barcodesequence of the second nucleic acid barcode molecule 916 can be coupledto the second particle 912 before and/or after partitioning theparticle. A lookup table (LUT) can be used to associate the shape,color, or a combination of shape and color (or other optical properties)of the particle (e.g., round or square bead) with the barcode sequence(e.g., first or second barcode sequence). For example, the firstparticle 908 with the round bead can be associated with the firstbarcode sequence of the first nucleic acid barcode molecule 914. Thesecond particle 912 with the square bead can be associated with thesecond barcode sequence of the second nucleic acid barcode molecule 916.Alternatively or in addition to, color may be used to differentiate thefirst particle 908 and the second particle 912.

With continued reference to FIG. 9, the first and second biologicalparticles can have different phenotypic properties, such as a circularshape of the cell and a square shape of the cell, respectively. Thefirst and the second partitions can be imaged to obtain a first opticalcharacterization and a second optical characterization of the partitionsin operations 918 and 920, respectively. The first and second opticalcharacterizations can include the physical characterization of theparticles and the phenotypic information of the biological particles.The biological particles (e.g., first biological particle) can eachcomprise a nucleic acid molecule. The nucleic acid molecule of the firstbiological particle 906 can be barcoded using the first barcode sequenceof the first nucleic acid barcode molecule 914 to generate a firstbarcoded nucleic acid molecule 922. The nucleic acid molecule of thesecond biological particle 910 can be barcoded using the second barcodesequence of the second nucleic acid barcode molecule 916 to generate asecond barcoded nucleic acid molecule 924. The first and second barcodednucleic acid molecules can be sequenced (e.g., together or separately)to generate a first nucleic acid sequence 926 and a second nucleic acidsequence 928, respectively, corresponding to the nucleic acid moleculesof the first and second biological particles and/or the first and secondbarcode sequences.

With continued reference to FIG. 9, a barcode sequence in a nucleic acidsequence can enable identification of the partition from which thebarcode sequence derives (e.g., the particle from which the barcodesequence derives) by electronically associating the nucleic acidsequence with physical and/or optical information of the partition. Theelectronic association can permit associating nucleic acid sequence withphenotypic information (e.g., shape of cell) of the biological particle(e.g., cell). The nucleic acid sequence can comprise genetic informationof the biological particle. For example, in operation 930, the firstbarcode sequence of the first barcode nucleic acid molecule 914 in thefirst nucleic acid sequence 926 can be used to identify the firstparticle 908 using a LUT. Identifying the first particle 908 can permitidentifying of the first biological particle 906 by using the firstoptical characterization 918. Further, the phenotypic information of thefirst biological particle 906 (e.g., circular cell) can be associatedwith the first nucleic acid sequence 926, in turn to genetic informationof the biological particle. Similarly, in operation 932, the secondbarcode sequence of the second nucleic acid barcode molecule 916 in thesecond nucleic acid sequence can be used to identify the second particle912 using a LUT. Identifying the second particle 912 can permitidentifying of the second biological particle 910 by using the secondoptical characterization 920. Further, the phenotypic information of thesecond biological particle 910 (e.g., square shape of cell) can beassociated with the second nucleic acid sequence 928, in turn to geneticinformation of the biological particle.

The present disclosure also provides a kit comprising a plurality ofparticles (e.g., as described herein). A kit may comprise a plurality ofparticles, which plurality of particles comprise (i) a plurality ofnucleic acid barcode molecules coupled thereto, wherein the plurality ofbarcode molecules comprise a plurality of barcode sequences. Theplurality of particles may also comprise (ii) a plurality of opticalbarcodes, wherein the plurality of optical barcodes comprise a pluralityof optical codes. Each particle of the plurality of particles maycomprise (i) a subset of the plurality of nucleic acid barcode moleculescoupled thereto and (ii) a subset of the plurality of optical barcodes.Barcode sequences of subsets of the plurality of nucleic acid barcodemolecules differ across particles of the plurality of particles, andoptical codes of subsets of the plurality of optical barcodes differacross the particles. Each subset of the subsets of the plurality ofoptical barcodes may comprise a single optical barcode or two or moreoptical barcodes. The plurality of optical barcodes may confer opticalproperties to the plurality of particles. Such optical properties may beselected from the group consisting of, for example, an absorbance, abirefringence, a color, a fluorescence characteristic, a luminosity, aphotosensitivity, a reflectivity, a refractive index, a scattering, or atransmittance of the particle or a component thereof.

As described elsewhere herein, optical barcodes may comprise one or morefluorescent dyes, nanoparticles, microparticles, or any combinationthereof. For example, optical barcodes may comprise a plurality ofnanoparticles, such as a plurality of quantum dots or Janus particles.In another example, optical barcodes may comprise a plurality offluorescent dyes, such as between 2-10 fluorescent dyes, such as 4fluorescent dyes. A plurality of fluorescent dyes may comprisefluorescent dyes having the same emission wavelength and differentintensities, such as between 2 and 50 different intensities, such as 20different intensities. Each subset of the subsets of the plurality ofoptical barcodes may comprise different optical codes. A plurality ofoptical barcodes may have associated optical intensities or frequenciesthat are distinct with respect to one another. A plurality of opticalbarcodes may comprise at least 1,000 different optical codes, such as atleast 10,000 different optical codes or at least 100,000 differentoptical codes.

As described elsewhere herein, nucleic acid barcode molecules within agiven subset of the subsets of the plurality of nucleic acid barcodemolecules may comprise identical barcode sequences. A plurality ofbarcode sequences may comprise at least 1,000 different barcodesequences, such as at least 10,000 different barcode sequences or atleast 100,000 different barcode sequences. A plurality of nucleic acidbarcode molecules may comprise a plurality of functional sequences(e.g., starter sequences, sequencing adapters, priming sequences, randomN-mers, capture sequences, etc.). Functional sequences may be configuredto interact with one or more target molecules, such as one or moredifferent types of target molecules (e.g., RNA molecules and DNAmolecules). In an example, a plurality of nucleic acid barcode moleculesattached to a given particle may each comprise an N-mer sequence (e.g.,a random N-mer sequence), which N-mer sequences are configured tointeract with a plurality of DNA molecules. In another example, aplurality of nucleic acid barcode molecules attached to a given particlemay each comprise a poly(T) sequence, which poly(T) sequences areconfigured to interact with a plurality of RNA molecules (e.g.,messenger RNA (mRNA) molecules). Functional sequences may be configuredto capture target molecules. For example, functional sequences may beconfigured to hybridize to target molecules to facilitate nucleic acidextension reactions.

The plurality of particles of a kit may comprise at least 10,000particles, such as at least 100,000 particles. The plurality ofparticles may comprise a plurality of beads, such as a plurality of gelbeads. The plurality of gel beads may comprise polyacrylamide polymers.The plurality of gel beads may each comprise a plurality of nucleic acidbarcode molecules releasably coupled thereto. For example, a first beadmay comprise a first plurality of nucleic acid barcode moleculesreleasably coupled thereto, and a second bead may comprise a secondplurality of nucleic acid barcode molecules releasably coupled thereto.Optical barcodes (e.g., optical materials such as fluorescent dyes) maybe included on surfaces of particles and/or within particles. In anexample, first particles of the plurality of particles are coupled tosecond particles of the plurality of particles, wherein the firstparticles comprise nucleic acid barcode molecules of the plurality ofnucleic acid barcode molecules and the second particles comprise opticalbarcodes of the plurality of optical barcodes.

Sample Partitioning

Provided herein are methods and systems for processing a nucleic acidmolecule from a cell. The methods can be useful for electronicallyassociating optical information of the cell with a nucleic acid sequenceof a nucleic acid molecule from the cell.

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of macromolecularconstituent contents of individual biological particles into discretecompartments or partitions (referred to interchangeably herein aspartitions), where each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion. A partition may comprise one or more otherpartitions.

A partition of the present disclosure may comprise biological particlesand/or macromolecular constituents thereof. A partition may comprise oneor more gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A cell bead can be a biologicalparticle and/or one or more of its macromolecular constituents encasedinside of a gel or polymer matrix, such as via polymerization of adroplet containing the biological particle and precursors capable ofbeing polymerized or gelled. Unique identifiers, such as barcodes, maybe injected into the droplets previous to, subsequent to, orconcurrently with droplet generation, such as via a microcapsule (e.g.,bead), as described further below. Microfluidic channel networks (e.g.,on a chip) can be utilized to generate partitions as described herein.Alternative mechanisms may also be employed in the partitioning ofindividual biological particles, including porous membranes throughwhich aqueous mixtures of cells are extruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can comprisedroplets of aqueous fluid within a non-aqueous continuous phase (e.g.,oil phase). The partitions can comprise droplets of a first phase withina second phase, wherein the first and second phases are immiscible. Avariety of different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual biologicalparticles to discrete partitions may in one non-limiting example beaccomplished by introducing a flowing stream of biological particles inan aqueous fluid into a flowing stream of a non-aqueous fluid, such thatdroplets are generated at the junction of the two streams. By providingthe aqueous stream at a certain concentration and/or flow rate ofbiological particles, the occupancy of the resulting partitions (e.g.,number of biological particles per partition) can be controlled. Wheresingle biological particle partitions are used, the relative flow ratesof the immiscible fluids can be selected such that, on average, thepartitions may contain less than one biological particle per partitionin order to ensure that those partitions that are occupied are primarilysingly occupied. In some cases, partitions among a plurality ofpartitions may contain at most one biological particle (e.g., bead, cellor cellular material). In some embodiments, the relative flow rates ofthe fluids can be selected such that a majority of partitions areoccupied, for example, allowing for only a small percentage ofunoccupied partitions. The flows and channel architectures can becontrolled as to ensure a given number of singly occupied partitions,less than a certain level of unoccupied partitions and/or less than acertain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying biological particles, cell beads, and/orgel beads that meet at a channel junction. Fluid may be directed flowalong one or more channels or reservoirs via one or more fluid flowunits. A fluid flow unit can comprise compressors (e.g., providingpositive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsulescarrying barcoded nucleic acid molecules (e.g., oligonucleotides)(described in relation to FIG. 2). The occupied partitions (e.g., atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe occupied partitions) can include both a microcapsule (e.g., bead)comprising barcoded nucleic acid molecules and a biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule that comprises an outer shell, layer or porous matrix inwhich is entrained one or more individual biological particles or smallgroups of biological particles. The microcapsule may include otherreagents. Encapsulation of biological particles may be performed by avariety of processes. Such processes combine an aqueous fluid containingthe biological particles with a polymeric precursor material that may becapable of being formed into a gel or other solid or semi-solid matrixupon application of a particular stimulus to the polymer precursor. Suchstimuli can include, for example, thermal stimuli (either heating orcooling), photo-stimuli (e.g., through photo-curing), chemical stimuli(e.g., through crosslinking, polymerization initiation of the precursor(e.g., through added initiators), or the like, and/or a combination ofthe above.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1, may be readily used in encapsulating cells as describedherein. In particular, and with reference to FIG. 1, the aqueous fluid112 comprising (i) the biological particles 114 and (ii) the polymerprecursor material (not shown) is flowed into channel junction 110,where it is partitioned into droplets 118, 120 through the flow ofnon-aqueous fluid 116. In the case of encapsulation methods, non-aqueousfluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid molecules todissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216along the channel segment 202 into junction 210. The plurality ofbiological particles 216 may be sourced from a suspension of biologicalparticles. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of biological particles 216.In some instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 1 micrometers 5 μm, 10 μm, 20μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500μm, 1 mm, or greater. In some cases, a bead may have a diameter of lessthan about 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 m, 250 m, 500 m, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 m, 30-75 m, 20-75 m, 40-85m, 40-95 m, 20-100 m, 10-100 m, 1-100 m, 20-250 m, or 20-500 m.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or a one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100pL, 50 pL, 20pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles's contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

In addition to the lysis agents co-partitioned with the biologicalparticles described above, other reagents can also be co-partitionedwith the biological particles, including, for example, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. In addition, inthe case of encapsulated biological particles, the biological particlesmay be exposed to an appropriate stimulus to release the biologicalparticles or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated biological particle to allow for the degradation ofthe microcapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of nucleic acid molecules (e.g.,oligonucleotides) from their respective microcapsule (e.g., bead). Inalternative aspects, this may be a different and non-overlappingstimulus, in order to allow an encapsulated biological particle to bereleased into a partition at a different time from the release ofnucleic acid molecules into the same partition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNAse, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutyn1-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114 115, 116, 117 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particle, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2). In someaspects, the unique identifiers are provided in the form of nucleic acidmolecules (e.g., oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, a single nucleic acid barcode sequence may beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). In some cases, the length of a barcodesequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 nucleotides or longer. In some cases, the length of a barcodesequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 nucleotides or longer. In some cases, the length of abarcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may becompletely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may beabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides orlonger. In some cases, the barcode subsequence may be at least about 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, a, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan \; \alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan \; \alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 μm, h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, a (not shown in FIG. 6)at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 10 shows a computer system1001 that is programmed or otherwise configured to (i) control amicrofluidics system (e.g., fluid flow), (ii) sort occupied dropletsfrom unoccupied droplets, (iii) polymerize droplets, (iv) imageindividual droplets to obtain optical information, (v) performsequencing applications, (vi) generate and maintain a library ofbarcoded nucleic acid molecules, (vii) analyze sequencing data, (viii)mapping sequencing data to a reference genome, and (ix) electronicallyassociate sequencing data to the individual droplet. The computer system1001 can regulate various aspects of the present disclosure, such as,for example, regulating polymerization application units for generatingparticles (e.g., beads) with different physical and/or opticalproperties, regulating fluid flow rate in one or more channels,regulating fluid flow to enable an imaging unit to image individualdroplets flowing through the channel and storing optical information ofthe droplets for further processing. The computer system 1001 can be anelectronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 1001 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1005, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1001 also includes memory or memorylocation 1010 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1015 (e.g., hard disk), communicationinterface 1020 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1025, such as cache, othermemory, data storage and/or electronic display adapters. The memory1010, storage unit 1015, interface 1020 and peripheral devices 1025 arein communication with the CPU 1005 through a communication bus (solidlines), such as a motherboard. The storage unit 1015 can be a datastorage unit (or data repository) for storing data. The computer system1001 can be operatively coupled to a computer network (“network”) 1030with the aid of the communication interface 1020. The network 1030 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1030 insome cases is a telecommunication and/or data network. The network 1030can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1030, in some cases withthe aid of the computer system 1001, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1001 tobehave as a client or a server.

The CPU 1005 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1010. The instructionscan be directed to the CPU 1005, which can subsequently program orotherwise configure the CPU 1005 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1005 can includefetch, decode, execute, and write back.

The CPU 1005 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1001 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1015 can store files, such as drivers, libraries andsaved programs. The storage unit 1015 can store user data, e.g., userpreferences and user programs. The computer system 1001 in some casescan include one or more additional data storage units that are externalto the computer system 1001, such as located on a remote server that isin communication with the computer system 1001 through an intranet orthe Internet.

The computer system 1001 can communicate with one or more remotecomputer systems through the network 1030. For instance, the computersystem 1001 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1001 via the network 1030.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1001, such as, for example, on thememory 1010 or electronic storage unit 1015. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1005. In some cases, thecode can be retrieved from the storage unit 1015 and stored on thememory 1010 for ready access by the processor 1005. In some situations,the electronic storage unit 1015 can be precluded, andmachine-executable instructions are stored on memory 1010.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1001, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1001 can include or be in communication with anelectronic display 1035 that comprises a user interface (UI) 1040 forproviding, for example, optical information of droplets, phenotypicinformation of biological particles (e.g., cell) results of sequencinganalysis and association of sequence data to the optical information.Examples of UIs include, without limitation, a graphical user interface(GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1005. Thealgorithm can, for example, obtain optical information of droplets,perform sequencing, perform sequence analysis, associate sequence datato the optical information, etc.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for processing a nucleic acid moleculeof a biological particle, comprising: (a) providing a plurality ofpartitions, wherein a partition of said plurality of partitionscomprises (i) a particle comprising a plurality of nucleic acid barcodemolecules, wherein a nucleic acid barcode molecule of said plurality ofnucleic acid barcode molecules comprises a barcode sequence, and (ii) abiological particle comprising said nucleic acid molecule; (b) usingsaid nucleic acid barcode molecule of said plurality of nucleic acidbarcode molecules and said nucleic acid molecule of said biologicalparticle to generate a barcoded nucleic acid molecule; (c) generating afirst data set comprising data identifying a nucleic acid sequence ofsaid barcoded nucleic acid molecule or a derivative thereof; and (d)using said first data set and a second data set comprising datacorresponding to one or more physical properties of said biologicalparticle to associate said one or more physical properties of saidbiological particle with said nucleic acid sequence.
 2. The method ofclaim 1, wherein said one or more physical properties of said biologicalparticle includes a size of said biological particle, a shape of saidbiological particle, a surface marker on said biological particle, aninclusion in said biological particle, a structure of an organelle insaid biological particle, a number of organelles in said biologicalparticle, a secretion or excretion with respect to said biologicalparticle, a localization of an organelle in said biological particle, ora combination thereof.
 3. The method of claim 1, wherein said biologicalparticle comprises a plurality of nucleic acid molecules.
 4. The methodof claim 3, wherein said plurality of nucleic acid molecules comprises aplurality of deoxyribonucleic acid (DNA) molecules or a plurality ofribonucleic acid (RNA) molecules.
 5. The method of claim 1, wherein saidnucleic acid molecule is a ribonucleic acid (RNA) molecule or adeoxyribonucleic acid (DNA) molecule.
 6. The method of claim 1, whereinsaid one or more physical properties comprises phenotypic information ofsaid biological particle.
 7. The method of claim 1, further comprisingoptically detecting said particle to generate a third data setcomprising data corresponding to one or more physical properties of saidparticle or said plurality of nucleic acid barcode molecules.
 8. Themethod of claim 7, wherein said one or more physical properties of saidparticle comprises a size, a shape, a circularity, a hardness, or asymmetry of said particle, or a combination thereof.
 9. The method ofclaim 7, wherein said one or more physical properties of said particlecomprises an optical property of said particle, which optical propertyof said particle comprises an absorbance, a birefringence, a color, afluorescence characteristic, a luminosity, a photosensitivity, areflectivity, a refractive index, a scattering, or a transmittance ofsaid particle.
 10. The method of claim 1, wherein nucleic acid barcodemolecules of said plurality of nucleic acid barcode molecules comprisebarcode sequences, which barcode sequences are identical.
 11. The methodof claim 1, wherein said plurality of partitions is a plurality ofdroplets.
 12. The method of claim 1, wherein said plurality ofpartitions is a plurality of wells.
 13. The method of claim 1, whereinsaid particle comprises one or more optical barcodes.
 14. The method ofclaim 13, wherein said plurality of nucleic acid barcode molecules ofsaid particle comprise said one or more optical barcodes.
 15. The methodof claim 13, wherein said one or more optical barcodes comprises afluorescent dye, a nanoparticle, a microparticle, or any combinationthereof.
 16. The method of claim 13, wherein said one or more opticalbarcodes has an associated optical intensity or frequency that isdistinct with respect to other optical barcodes of other partitions ofsaid plurality of partitions.
 17. The method of claim 1, wherein saidone or more physical properties of said biological particle areidentified by imaging said partition using (i) bright field microscopy,(ii) fluorescence microscopy, (iii) phase contrast microscopy, (iv)multispectral microscopy, or (v) polarization microscopy.
 18. The methodof claim 1, wherein said particle is a bead.
 19. The method of claim 18,wherein said bead is a gel bead.
 20. The method of claim 18, whereinsaid plurality of nucleic acid barcode molecules is releasably coupledto said bead.
 21. The method of claim 1, further comprising, subsequentto (a), imaging said partition to optically detect said biologicalparticle, thereby generating said data corresponding to said one or morephysical properties of said biological particle, wherein said one ormore physical properties are one or more optical properties.
 22. Themethod of claim 1, further comprising, prior to (a), imaging saidbiological particle to generate said data corresponding to said one ormore physical properties of said biological particle, wherein said oneor more physical properties are one or more optical properties.
 23. Themethod of claim 1, wherein said particle is coupled to another particlecomprising one or more optical barcodes.
 24. The method of claim 1,wherein (a)-(d) are repeated for additional partitions among saidplurality of partitions, wherein said additional partitions eachcomprise (i) an additional plurality of nucleic acid barcode molecules,wherein a nucleic acid barcode molecule of said additional plurality ofnucleic acid barcode molecules comprises an additional barcode sequencedifferent than said barcode sequence, and (ii) an additional biologicalparticle comprising an additional nucleic acid molecule, and whereinsaid partition and said additional partitions comprise a combination ofparticles comprising molecular barcodes and particles comprising opticalbarcodes, which combination is different across said partition and saidadditional partitions.
 25. The method of claim 1, wherein (a)-(d) arerepeated for additional partitions among said plurality of partitions,wherein said additional partitions each comprise (i) an additionalplurality of nucleic acid barcode molecules, wherein a nucleic acidbarcode molecule of said additional plurality of nucleic acid barcodemolecules comprises an additional barcode sequence different than saidbarcode sequence, and (ii) an additional biological particle comprisingan additional nucleic acid molecule, which additional partitionscomprises 1,000 partitions, and wherein said additional partitionscomprise a plurality of particles comprising nucleic acid barcodemolecules comprising 1,000 barcode sequences, wherein said 1,000 barcodesequences are different across said partition and said additionalpartitions.
 26. The method of claim 1, wherein said biological particleis a cell.
 27. The method of claim 1, wherein said biological particlecomprises a cell, or one or more components thereof, in a polymeric orgel matrix.
 28. The method of claim 1, further comprising associating aprotein of said biological particle with said one or more physicalproperties of said biological particle with said nucleic acid sequence.29. The method of claim 1, further comprising associating one or moreribonucleic acid (RNA) sequences or one or more deoxyribonucleic acid(DNA) sequences of said biological particle with said one or morephysical properties of said biological particle with said nucleic acidsequence.
 30. The method of claim 29, wherein said one or more DNAsequences comprise epigenetic information or chromatin information.